Salinomycin-loaded lipid-polymer nanoparticles with anti-CD20 aptamers selectively suppress human CD20+ melanoma stem cells.

Melanoma is the deadliest type of skin cancer. CD20+ melanoma stem cells (CSCs) are pivotal for metastasis and initiation of melanoma. Therefore, selective elimination of CD20+ melanoma CSCs represents an effective treatment to eradicate melanoma. Salinomycin has emerged as an effective drug toward various CSCs. Due to its poor solubility, its therapeutic efficacy against melanoma CSCs has never been evaluated. In order to target CD20+ melanoma CSCs, we designed salinomycin-loaded lipid-polymer nanoparticles with anti-CD20 aptamers (CD20-SA-NPs). Using a single-step nanoprecipitation method, salinomycin-loaded lipid-polymer nanoparticles (SA-NPs) were prepared, then CD20-SA-NPs were obtained through conjugation of thiolated anti-CD20 aptamers to SA-NPs via a maleimide-thiol reaction. CD20-SA-NPs displayed a small size of 96.3 nm, encapsulation efficiency higher than 60% and sustained drug release ability. The uptake of CD20-SA-NPs by CD20+ melanoma CSCs was significantly higher than that of SA-NPs and salinomycin, leading to greatly enhanced cytotoxic effects in vitro, thus the IC50 values of CD20-SA-NPs were reduced to 5.7 and 2.6 µg/mL in A375 CD+20 cells and WM266-4 CD+ cells, respectively. CD20-SA-NPs showed a selective cytotoxicity toward CD20+ melanoma CSCs, as evidenced by the best therapeutic efficacy in suppressing the formation of tumor spheres and the proportion of CD20+ cells in melanoma cell lines. In mice bearing melanoma xenografts, administration of CD20-SA-NPs (salinomycin 5 mg·kg-1·d-1, iv, for 60 d) showed a superior efficacy in inhibition of melanoma growth compared with SA-NPs and salinomycin. In conclusion, CD20 is a superior target for delivering drugs to melanoma CSCs. CD20-SA-NPs display effective delivery of salinomycin to CD20+ melanoma CSCs and represent a promising treatment for melanoma.

[Importance of re-examinations in the diagnosis of benign paroxysmal positional vertigo].

OBJECTIVE: To evaluate the importance of re-examinations in the diagnosis of benign paroxysmal positional vertigo (BPPV) and its clinical characteristics. METHODS: The medical records of 288 BPPV patients treated during January 2009 to December 2010 were reviewed and analyzed. The definite diagnosis was established by re-examinations. RESULTS: Among them, 39 (13.5%) were diagnosed by re-examinations or return visits. And 25 patients received a correct diagnosis on the first visit and 14 were diagnosed on return visits. Their ages, gender distribution and the times of particle-repositioning maneuver were the same as the overall profiles. The rate of horizontal semicircular canal BPPV was higher in the 39 re-examined patients than the overall rate (46.15% vs 22.22%). And the latencies decreased during the second examination in 18 patients. CONCLUSION: Re-examinations and return visits are useful for diagnostic clarifications in the suspected BPPV patients.

Gut and Cutaneous Microbiome Featuring Abundance of Lactobacillus reuteri Protected Against Psoriasis-Like Inflammation in Mice.

BACKGROUND: Psoriasis is a chronic autoinflammatory skin disease, and its aetiology remains incompletely understood. Recently, gut microbial dysbiosis is found to be tightly associated with psoriasis. OBJECTIVE: We sought to reveal the causal role of gut microbiota dysbiosis in psoriasis pathogenesis and investigate the protective effect of healthy commensal bacteria against imiquimod -induced psoriasis-like skin response. METHODS: By using fecal microbial transplantation (FMT), 16S rRNA gene-based taxonomic profiling and Lactobacillus supplement, we have assessed the effect of FMT from healthy individuals on psoriasis-like skin inflammation and associated immune disorders in imiquimod-induced psoriasis mice. RESULTS: Here, by using psoriasis mice humanized with the stools from healthy donors and psoriasis patients, the imiquimod-induced psoriasis in mice with psoriasis patient stool was found to be significantly aggravated as compared to the mice with healthy donor stools. Further analysis showed fecal microbiota of healthy individuals protected against Treg/Th17 imbalance in psoriasis. Moreover, we found the gut and skin microbiome in mice receipted with gut microbiota of healthy individuals (HD) differed from those of mice receipted with gut microbiota of psoriasis patients (PSD). 16S rRNA sequencing revealed that Lactobacillus reuteri was greatly enriched in fecal and cutaneous microbiome of HD mice as compared to PSD mice. Intriguingly, supplement with Lactobacillus reuteri was sufficient to increase the expression of anti-inflammatory gene IL-10, reduce Th17 cells counts and confer resistance to imiquimod-induced inflammation on the mice with gut microbiota dysbiosis. CONCLUSION: Our results suggested that the gut microbiota dysbiosis is the potential causal factor for psoriasis and the gut microbiota may serve as promising therapy target for psoriasis patients.

[High-frequency rotation sensation function damage of the crista of the horizontal semicircular canal induced by gentamicin and its morphological basis.].

The goal of the present study was to explore high-frequency rotation sensation function damage of the crista of the horizontal semicircular canal induced by gentamicin and its morphological basis. The guinea pigs were randomly divided into four groups (group 1, 2, 3 and control group, n=20). The animals of treated groups received gentamicin subcutaneously (50 mg/kg per day) for 1 week (group 1), 2 weeks (group 2) and 3 weeks (group 3), respectively. The animals of control group were administered with equal volume of saline subcutaneously. Videonystagmography (VNG) evoked by ice water or high-frequency rotations, and vestibular evoked potential (VsEP) evoked by low- and high-frequency rotations were recorded after the administration. After VNG and VsEP were examined, the cristae of the horizontal semicircular canals were prepared for scanning and transmission electron microscopy. The results are as follows: (1) In VNG examination, after ice water stimulation, no obvious nystagmus was observed in the animals of group 3, and there was no significant difference in nystagmus frequency and duration among group 1, 2 and control group (P>0.05). After high- frequency rotations, no obvious nystagmus was yet observed in the animals of group 3. There were significant differences in the nystagmus frequency and duration of the VNG waves between the experimental group 2 and control group (P<0.01), and no difference was found between group 1 and control group (P>0.05). (2) In VsEP examination, there was no significant difference in the parameters of VsEP among group 1, 2 and control group (P>0.05), and VsEP was not observed in group 3 after low-frequency rotations. After high- frequency rotations, compared with control group, the latencies and amplitudes of P1 and P2 in group 2 significantly decreased (P<0.01), while no changes were found in group 1 (P>0.05). Group 3 had no response of VsEP to high-frequency rotations. (3) Electron microscopy was used to observe the crista hair cells of the four groups. In control group, the hair cells were normal. In group 1, almost normal appearance with slight edema of the hair cells was shown. In group 2, some hair cells in the central top of the crista necrosed, but other hair cells in the peripheral area showed normal appearance. In group 3, vestibular hair cells were entirely impaired or necrosis. These results show gentamicin selectively induces high-frequency rotation sensation function damage of the crista of the horizontal semicircular canal. The damage is concentrated on the central top area and then subsequently extended to the peripheral area of vestibular crista. It can be inferred that the hair cells in the central top area of the crista are involved in high-frequency rotation vestibular perception.

What is the most relevent factor causing pain during ALA-PDT? A multi-center, open clinical pain score research trial of actinic keratosis, acne and condylomata acuminata.

BACKGROUND: To date, it has been reported that the intrinsic factors(lesions location, lesions area, disease tynpes) and extrinsic factors(fluence rate) contribute to the pain during 5-aminolevulinic acid photodynamic therapy (ALA-PDT). But there are few studies on pain during ALA-PDT and lack of sufficient clinical evidence related to the pain intensity. OBJECTIVE: To investigate pain intensity and its relative factors during ALA-PDT and to provide clinical implication. METHODS: The pain numeric rating scale (PNRS) score was used to evaluate the patients' pain intensity at different times during ALA-PDT irradiation from 0 to 10 min during treatment. Gender, age, lesions location, lesions area, ALA concentration and fluence rate were recored. RESULTS: The trial enrolled 274 patients in total, including 118 acne patients (in face), 30 actinic keratosis(AK)patients(in face), 126 Condylomatata acuminate patients(in genitalia). The average pain score in PDT was highest in the patients with actinic keratosis(7.3 ± 0.7), and that of condylomata acuminata was the lowest (4.5 ± 1.1) (p < 0.05). The highest pain score in patients with AK, acne and condylomata acuminata was 8, 6 and 6 respectively which occurred at 4 min, 4 min and 6 min respectively. The pain score of males was higher compared with females in all of the three diseases (p < 0.05). The pain score of facial diseases (5.6 ± 1.2) was higher than that of the genitalia (4.5 ± 1.1) (p < 0.05). The lesions area was positively correlated with the pain score (p < 0.05). In facial diseases, the pain score of patients with high fluence rate (7.3 ± 0.7) was higher than patients with low fluence rate (5.1 ± 0.9) (p < 0.05). CONCLUSIONS: Intrinsic and extrinsic factors both correlate with pain during PDT. Intrinsic factors are difficult to change, so extrinsic factors are the key point to control. We can reduce the fluence rate and extend the treatment time, relieving pain intensity while still ensuring equivalent efficacy.

Evaluation of the otolith function using c/oVEMPs in patients with Ménière's disease.

BACKGROUND: Cervical and ocular vestibular evoked myogenic potentials (c/oVEMPs) reflect otolith function. Up-to-date, there are no published reports on the systemic evaluation of otolith function in Ménière's Disease (MD) nor are there any reports on the differences in VEMPs between patients with early and late stage MD. The aim of this study was to evaluate the difference in c/oVEMPs between patients with MD and normal controls, as well as between patients with early and late stage MD. METHODS: Thirty patients with unilateral MD and thirty healthy subjects (as normal controls) were prospectively enrolled. c/oVEMPs using 500 Hz tone-burst stimuli were performed. VEMP tests were repeated 3 times on each subject to ensure reliability and reproducibility of responses. VEMPs were defined as present or absent. Abnormal VEMP was defined by lack of VEMP response. RESULTS: In the control group, abnormal cVEMPs and oVEMPs responses were detected in 6.67 and 3.34 % respectively. In MD patients (20 with early stage MD [ES-MD], 10 with late stage MD [LS-MD]), abnormal cVEMPs and oVEMPs responses were detected in 40 and 16.7 % respectively. More patients with MD showed abnormal responses in c/oVEMPs as compared to the control group (p < 0.05). cVEMPs was more often abnormal as compared to oVEMPs in MD patients (p < 0.05). There was a significant difference in abnormal cVEMP responses between ES-MD patients (25 %) and LS-MD patients (70 %) (p < 0.05). Difference in abnormal oVEMP responses (ES-MD, 5 %; LS-MD, 40 %) was significant (p < 0.05). CONCLUSION: An increased occurrence of abnormal c/oVEMP recordings appeared in MD patients, possibly as a result of hydrops of the otolith. cVEMPs were more often abnormal in MD patients as compared to oVEMPs, suggesting that saccular dysfunction may be more common than utricular dysfunction. Furthermore, o/cVEMP abnormalities in the LS-MD group were significantly higher than those in the ES-MD group, suggesting the trend that otolith damage is gradually increasing with the aggravation of cochlear injury in MD.

Degree of sigmoid sinus compression and the symptom relief using magnetic resonance angiography in venous pulsating tinnitus.

OBJECTIVES: To show that mechanical compression of sigmoid sinus is effective for treatment of pulsatile tinnitus caused by sigmoid sinus enlargement, and to evaluate the relationship between the compression degree of sigmoid sinus and the tinnitus symptom relief using magnetic resonance angiography. METHODS: Medical records of twenty-four patients who were diagnosed with venous tinnitus caused by sigmoid sinus enlargement and underwent mechanical compression of sigmoid sinus were reviewed between April 2009 and May 2013. All these patients received computed tomography and magnetic resonance venography study before undergoing surgery and were followed for at least 4 months. RESULTS: Twenty-three patients felt relief from tinnitus three months after the surgery, and the cross-sectional area of the sigmoid sinus on the tinnitus side was compressed approximately by half (46%-69%) after the surgery. There were 4 patients whose tinnitus suddenly disappeared while lying on the operating table before operation, which may be a result of the patient's emotional tension or postural changes from standing. One of the four patients felt no relief from tinnitus after the surgery, with the cross-sectional area of the sigmoid sinus only compressed by 30%. And two patients of them had a recurrence of tinnitus about 6 months after the surgery. Seven patients had sigmoid sinus diverticula, and tinnitus would not disappear merely by eliminating the diverticulum until by compressing the sigmoid sinus to certain degree. There were 3 minor complications, including aural fullness, head fullness and hyperacusis. The preoperative low frequency conductive and sensorineural hearing loss of 7 subjects subsided. CONCLUSION: Mechanical compression of sigmoid sinus is an effective treatment for pulsatile tinnitus caused by sigmoid sinus enlargement, even if it might be accompanied by sigmoid sinus diverticulum. A compression degree of sigmoid sinus about 54% is adequate for the relief of tinnitus symptom. Cases in which patients' tinnitus suddenly disappeared before the surgery might be excluded to improve the efficacy of surgery.

Spontaneous healing of various tympanic membrane perforations in the rat.

OBJECTIVES: To investigate the spontaneous healing process of various types of perforation and the location of the epithelial generation center in the tympanic membrane of rats. MATERIAL AND METHODS: Various types of perforation were made in the ears of 50 rats and the healing process was observed using light and electron microscopy. RESULTS: Epithelia hyperplasia could not be seen at the edge of the perforations, but occurred in the annulus and handle of malleus regions during the early stage of healing. There was no correlation between the healing time and the size of the perforations. When one ear was perforated, no changes were observed in the intact ear on the other side. The epithelium did not migrate into the tympanic cavity in the posterior marginal perforations. All the perforations healed, although the manubria of the malleus were damaged. CONCLUSIONS: The perforations healed by means of epithelial migration. The epithelial generation center was located near the annulus and the handle of the malleus, and therefore protection of these two regions is very important in middle ear surgery.

[Effect of sigmoid sinus plasty in sigmoid sinus original pulsatile tinnitus].

OBJECTIVE: To discuss the diagnosis and management of venous original pulsatile tinnitus associated with sigmoid sinus. METHODS: A retrospective study was conducted on 12 patients who were diagnosed with venous original pulsatile tinnitus associated with sigmoid sinus, and treated with sigmoid sinus constriction surgery. The diagnostic evidences for venous original pulsatile tinnitus associated with sigmoid sinus were re-evaluated, the pulsatile tinnitus improvements and MRV study results before and after surgeries associated with sigmoid sinus were compared. RESULTS: Eleven patients got relief of tinnitus within three months after the surgeries, while one patient had no relief. There were ten patients underwent MRV study, the cross-sectional area of the sigmoid sinus in the healthy side was about two times in the tinnitus side. Constriction sigmoid sinus was performed on the twelve patients. The cross-sectional area of the sigmoid sinus of relieved tinnitus patients were compressed by forty-six percent to eighty-three percent. None of the cases complained of any serious complications. CONCLUSIONS: Sigmoid sinus constriction is an available therapy for pulsatile tinnitus at present. More cases and longer follow-up are necessary to evaluate its treatment effect accurately.

Narrow-band UVB radiation promotes dendrite formation by activating Rac1 in B16 melanoma cells.

Melanocytes are found scattered throughout the basal layer of the epidermis. Following hormone or ultraviolet (UV) light stimulation, the melanin pigments contained in melanocytes are transferred through the dendrites to the surrounding keratinocytes to protect against UV light damage or carcinogenesis. This has been considered as a morphological indicator of melanocytes and melanoma cells. Small GTPases of the Rho family have been implicated in the regulation of actin reorganization underlying dendrite formation in melanocytes and melanoma cells. It has been proven that ultraviolet light plays a pivotal role in melanocyte dendrite formation; however, the molecular mechanism underlying this process has not been fully elucidated. The effect of small GTPases, such as Rac1 and RhoA, on the morphology of B16 melanoma cells treated with narrow-band UVB radiation was investigated. The morphological changes were observed under a phase contrast microscope and the F-actin microfilament of the cytoskeleton was observed under a laser scanning confocal microscope. The pull-down assay was performed to detect the activity of the small GTPases Rac1 and RhoA. The morphological changes were evident, with globular cell bodies and increased numbers of tree branch-like dendrites. The cytoskeletal F-actin appeared disassembled following narrow-band UVB irradiation of B16 melanoma cells. Treatment of B16 melanoma cells with narrow-band UVB radiation resulted in the activation of Rac1 in a time-dependent manner. In conclusion, the present study may provide a novel method through which narrow-band UVB radiation may be used to promote dendrite formation by activating the Rac1 signaling pathway, resulting in F-actin rearrangement in B16 melanoma cells.

Assessment of middle ear effusion and audiological characteristics in young children with adenoid hypertrophy.

BACKGROUND: Otitis media with effusion is a highly concurrent disease in young children with adenoid hypertrophy. The aim of this study was to assess the middle ear effusion and audiological characteristics in children with adenoid hypertrophy and compare the various assessment methods. METHODS: Two hundred and seven candidates who were to undergo adenoidectomy were analyzed using otoscopy, tympanometry, air-conduction auditory steady-state responses (AC-ASSR), and computerized tomography (CT) before adenoidectomy. RESULTS: About 73.4% (304/414) of ears were confirmed to have middle ear effusion (MEE) by otoscopy; 75.4% (312/414) of ears revealed MEE by CT. CT scan correctly predicted all the myringotomy results, giving 100% accuracy on the diagnosis of MEE. Additionally, CT revealed two children with inner ear malformations. Type B tracing tympanogram provided a sensitivity of 91.7% and a specificity of 92.2%. Type C tympanogram with peak pressure < -200 daPa indicated effusion; type C tympanogram having acoustic stapedius reflex could exclude MEE. We excluded the AC-ASSR results of the 4 ears with malformation; 54.4% (223/410) of ears were confirmed of hearing loss. Furthermore, 5.2% (16/310) of the ears with MEE suffered from severe to profound hearing loss. The average threshold level in the 0.25 kHz frequency of children was found to have poorer hearing thresholds than those in the 0.5, 1, 2, and 4 kHz (P < 0.001) frequencies; 29.7% (92/310) of ears with MEE were regarded as normal hearing level. About 55.8% (173/310) of ears with MEE were classified as having slight-mild hearing loss. CONCLUSIONS: The practitioners should pay much attention to the middle ear condition and be aware of a possible development of severe to profound hearing loss during the course of MEE in young children with adenoid hypertrophy. CT scan is good for the assessment of MEE before ventilation tube insertion.

Cutaneous neuroendocrine carcinoma of the external auditory canal: a case report and review of the literature.

Cutaneous neuroendocrine carcinoma (cNEC) is rarely seen in the external ear. In this paper, we newly describe a patient with cNEC in his right external auditory canal, followed by a further discussion on the clinical features, diagnosis, and treatments of cNEC of the external ear. A review of the literature showed that cNEC of the external auditory canal generally presents as asymptomatic and that pathology yields the most confirmative diagnosis. A wide resection with adjuvant radiotherapy and chemotherapy is recommended. The overall prognosis of this condition is poor.

Frequency perception and cell injury in the semicircular canal caused by gentamicin.

OBJECTIVE: The frequency characteristics of the vestibular organ have gained notice in recent years, but the morphologic basis was unknown. This study investigated the gentamicin-induced damage of frequency-selective perception of the horizontal semicircular canal and its morphologic basis. METHODS: Eighty guinea pigs were randomly divided into four groups, one control group and three experimental groups. The experimental animals received gentamicin subcutaneously for 1 to 3 weeks. Short-latency vestibular evoked potentials evoked by 0.5 and 10 Hz step rotation stimuli following drug administration were recorded, and then the crista ampullaris of the horizontal semicircular canals was investigated by scanning and transmission electron microscopy. RESULTS: Damage to hair cells of the crista ampullaris is concentrated at the apex area first and then extends to the peripheral area of the vestibular crista ampullaris when the gentamicin administration time increased. When only the hair cells at the apex area are damaged, the high-frequency (10 Hz) rotation perception of the crista ampullaris of the horizontal semicircular canal was injured, but perceptions to 0.5 Hz step rotation stimulation remained normal. CONCLUSION: Gentamicin mainly affects the high-frequency perception function of the crista ampullaris of the horizontal semicircular canal. The hair cells at the central apex area of the crista ampullaris might be responsible for high-frequency rotation perception function.

[Langerhans cell histiocytosis of the temporal bone: 22 cases analysis].

OBJECTIVE: To analyze the clinical manifestations, diagnosis, therapy and prognosis of Langerhans cell histiocytosis (LCH) of the temporal bone. METHODS: Twenty-two patients with LCH of the temporal bone in our hospital were retrospectively summed up from January 1994 to January 2008. Following up 1 - 15 years, their clinical manifestations, diagnosis, therapy and prognosis were studied. Survival analysis and disease free survival Log-rank test were used. RESULTS: Among 22 patients, 1 case belonged to the multisystem high risk group, 3 cases to the multisystem low risk group, and 18 cases to the single system group. The clinical characters were primarily the ear presentations, CT of the temporal bones showed extensive osteolytic destructions. The misdiagnosis rate was 72.7%. Twenty-two cases were received different combined modality therapies. Following up 1 - 15 years, 21 cases were survival and 1 case dead (4.5%), while 5 cases showed residual of insipidus, dwarfism, epilepsy or unilateral serious hearing loss (22.7%). For disease free survival curve, there was a significant difference between the multisystem group and the single system group (chi(2) = 5.87, P < 0.05). CONCLUSIONS: As LCH of the temporal bone, the single system cases are predominant. The ear area clinical manifestations are primary. This disease is easily misdiagnosed. The therapy selection should rely on the involved systems of the disease. The prognosis of the single system group is much better than the multisystem group.

[Normative data and clinical practice of sinusoidal harmonic acceleration test].

OBJECTIVE: To establish the normative data of sinusoidal harmonic acceleration test (SHAT) and evaluate the value of clinical application. METHODS: One hundred and twenty normal persons, 21 Meniere's disease patients, 2 bilateral vestibular peripheral lesion patients, 15 unilateral vestibular peripheral lesion patients and 10 central lesion patients were tested with SHAT. RESULTS: The calibration time was longer in the older persons. The phase decreased with the frequency, and the gain increased with the frequency, but the reliability of the re-test of the phase was better in the lower frequency. The results of the 7 Meniere's disease patients without symptoms were normal, 14 patients post-attack revealed abnormal, 11 had phase abnormal, 3 had gain decrease, 10 revealed asymmetry and spontaneous nystagmus simultaneously, 8 patients had two parameters abnormal. The unilateral vestibular peripheral lesion patients showed 73% (11/15) phase abnormal, 67% (10/15) gain decrease and 40% (6/15) asymmetry, while 5/6 asymmetry patients had spontaneous nystagmus. The gain of the bilateral vestibular peripheral lesion patients showed severe decrease. The abnormal rate in central lesion patients were 70% (7/10) in phase, 20% (2/10) in gain and 40% (4/10) in asymmetry, but the patients with asymmetry had no spontaneous nystagmus. CONCLUSIONS: The normal range of the phase, gain and asymmetry of SHAT is in narrow bandwidth. The phase is the most important abnormal sign.

[Spontaneous healing of various types of rat tympanic membrane perforation].

OBJECTIVE: To investigate the spontaneous healing process of various types of rat tympanic membrane perforation, study the perforation healing mechanism. METHODS: Fifty rats were divided into five groups in randomization, and performed perforations phi = 2.5 mm, 1 mm, respectively in the center of tympanic membrane pars tensa, posterior marginal perforation (phi = 1.5 mm) in pars tensa, the handle of malleus cauterized with chromic acid after tympanic membrane 3/4 excision, and perforation (phi = 1.5 mm) in the center of pars flaccid, The histology of the perforations healing process was observed with light microscope. RESULTS: The epithelia retracted in the early stage after perforation. The epithelia near the annulus and manubrium showed hyperplasia. The epithelia hyperplasia also appeared the intact annulus region remote to the perforation. The middle connective tissue layer reacted later than the epithelial layer. The defects were closed first by the accumulation of the epithelial cells. The difference of healing time had no statistics significance between the big and small perforations. One ear perforated, but the other side intact ear appears no change. The epithelia didn't migrate into the tympanic cavity in the posterior marginal perforations. All the perforations healed after the manubriums of the malleus were damaged. Perforations in pars flaccid healed more quickly. CONCLUSIONS: The normal migration of the epithelia of the tympanic membrane played an important role in the healing of perforation. The generation center was located near tympanic annulus and manubrium of the malleus, so the protection of these two regions was very important in the middle-ear surgery.

[Epidermal stem cells in the tympanic membrane].

OBJECTIVE: To investigate the distributions of epithelial stem cells in the tympanic membrane and the growth characteristics of cultured epithelial cells from different region of tympanic membrane, and to establish culture techniques of stem cells in tympanic membrane. METHODS: Four young rats and four adult SD rats were used to observe normal tympanic membrane. The other 28 rats were performed 2 mm size perforations in pars tensa. These animals were sacrificed at different periods after perforation. The tympanic membranes were cut in cryostat sections for immunohistochemistry of cytokeratin 19 and integrin beta1. Thirty tympanic membranes of rats were treated with Mitomycin C to damage the mucosal surface, then divided into two parts: the annulus region and center region of pars tensa, cultured in medium with high amount of epidermal growth factor and low amount of calcium. RESULTS: The immunostaining cells of cytokeratin 19 and integrin beta1 were displayed in both the handle of malleus and annular regions, but there were no staining positive cells in the intermediate region of pars tensa. The positive cells distribution had no significant difference between adult and infancy rats. In the pars flaccid, the positive cells scattered in the basal layer. The positive cells increased after perforation in the annulus and handle of malleus region, but no immunostaining cells were found at the edge of perforation. The redouble time of the culture cells from the annulus region was shorter than the center of pars tensa. The cells adherent within 1 hour formed larger and more colonies, and contained more positive cells. CONCLUSION: The epithelial stem cells in tympanic membrane were located in both the handle of malleus and annular regions, but no stem cells could be found in the intermediate region of pars tensa. The stem cells of tympanic membrane can be simply purified according to the adherent time.